Nanopore Detection of miRNAs Using p19

When: Thursday, April 05, 2012 at 12:00 pm
Where: DA 114
Speaker: Dr. Larry McReynolds
Organization: New England Biolabs
Sponsor: Biophysical Group Meeting

Detection of microRNAs often requires labeling, ligation or amplification of the RNA.  We have developed a novel method that utilizes the high affinity binding of the plant viral protein p19. The p19 protein binds complementary dsRNA of 19-22 bp with high affinity resulting in over 100,000 enrichment of dsRNA from total cellular RNA.  It does not bind ssRNA. The p19 fusion protein, bound to chitin magnetic beads, was used to isolated a specific miRNA:probe duplex.  The eluted RNA was detected with an ultrathin 3nm silicon nitride nanopore that can discriminate short siRNAs from tRNA and dsDNA.  The use of ultrathin 7 nm thick membranes increased the amplitude of the signal, which enabled detection of the unlabeled miRNA.  Using this device we detected femtomolar amounts of miR-122 isolated from total rat liver RNA with p19.